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One of the human genetic diseases molecularly well characterized and suited for gene therapy is cystic fibrosis CF. CF is the most common autosomal recessive disease in the white population and affects approximately 70, individuals worldwide Rosenecker et al. Cloning of the CF transmembrane conductance regulator CFTR gene and a better understanding of the disease pathophysiology, and promising proof-of-principle preclinical studies on CFTR gene transfer, led to the initiation of human clinical trials.

Despite encouraging safety profiles in phase I and phase II trials, none of the primary end points changed significantly Wagner et al. A major limitation for this is the lack of efficient vector transduction to airway epithelial cells through the apical surface. Thus, it was apparent that molecular alterations to enhance AAV transduction to airway epithelium would advance their clinical utility.

Several alternative approaches are being attempted with AAV vectors to achieve high-efficiency transduction of target tissues, such as airway epithelium; these approaches include the use of a shortened AAV cassette Ostedgaard et al. Additional strategies to increase the permeability of the apical surface of the airway epithelium to AAV, using chemicals and a combination of transduction-enhancing compounds after vector transduction, have resulted in significant augmentation of gene expression in airway epithelium Duan et al.

Thus, it is apparent that further advancements in targeted transduction of AAV vectors to airway epithelium will positively impact on their clinical utility. Genetic capsid modification remains a viable alternative to enhance AAV gene transfer to target cells through alternative cellular receptors. However, approaches employing this strategy for CF gene therapy remain unexplored.

To this end, we developed rAAV with genetic capsid modification to include a panel of putative airway epithelium-specific ligand sequences in the GH loop of AAV2 capsids and tested them in polarized human airway epithelial cells. To achieve polarization, 10 6 Calu-3 cells were seeded on 6.

The plasmid pSP62, containing the rep and cap genes of AAV2, was digested with the restriction enzyme Apa I to generate a bp fragment between nucleotide junctions and in the AAV2 capsid region. Sequences of the oligonucleotides used for site-directed mutagenesis for tested ligands are as follows:. Sequences underlined in M1 correspond to regions in the AAV2 capsid flanking the ligand insert. This sequence is common to all six mutants. The PCR products were transformed into XL-Blue competent cells and positive clones were identified by restriction digestion and automated sequencing, using the T7 primer.

Positive clones were again confirmed by restriction digestion and automated sequencing. Particle titer of the purified virus was determined by quantitative slot-blot analysis and real-time PCR as previously described Ren et al. The membranes were probed with the AAV2 capsid monoclonal antibody B1, diluted , followed by the addition of horseradish peroxidase-conjugated goat antimouse IgG secondary antibody diluted to Approximately 10 5 cells were seeded per well in well tissue culture plates.

All assays were performed in triplicate. Statistical significance among means was determined by Student t test. During the initiation of this study, a few peptide ligands had been identified by in vitro phage display with high-affinity binding to human airway epithelium or to alternative receptors highly expressed on airway epithelium.

These two peptide sequences were found to bind with high affinity to the human transferrin receptor Jost et al. Ligands of apically located receptors on human airway epithelium, which bind the target cells more specifically and with higher affinity, are needed for most gene therapy applications. Either peptide sequence could mediate transduction from the apical or basolateral side of polarized 16HBE14o- cells.

Similar to Calu-3, this cell line forms polarized airway epithelium expressing the morphological characteristics of respiratory cells in vivo. The synthesized peptide motif THALWHT was coupled to a cationic DNA-binding moiety and was shown to mediate efficiently targeted gene delivery into 16HBE14o- cells, indicating the possibility of similar results when applied to other vector systems.

We created six different capsid mutants containing the previously described peptide sequences by site-directed mutagenesis. On positive sequence confirmation of the capsid open reading frame ORF of AAV-2 mutants, cells were transfected and subjected to Western blot analysis. The results of Western blot analysis, shown in Fig. B1 antibody, which recognizes linear epitope IGTRYLTR in the C-terminal region of all three capsid proteins, was used to confirm that the plasmids containing genetically modified capsids expressed capsid proteins of the appropriate size and ratio Wistuba et al.

A notable increase in the molecular masses of mutant capsid proteins further confirmed that heterologous peptide ligands were successfully incorporated into VP-1, VP-2, and VP Western blot analysis of cell lysates containing wild-type wt and mutant rAAV capsids and transgene expression in cells with rAAV-luc encapsidated in wild-type or mutant capsids.

Luciferase activity was determine from cell lysates and expressed as relative light units RLU , normalized to the protein content of each cell lysate. After this, we packaged rAAV encoding luciferase in wild-type and the six mutant capsids. Purification of the recombinant vector encapsidated in wild-type and the six different mutant capsids was done by heparin column affinity chromatography.

On the basis of the heparin-binding region in the AAV capsid, amino acids R and R, it was thought that ligand insertion between amino acids and may abolish heparin binding due to the proximity of the insertion site to the heparan sulfate proteoglycan HSPG -binding region Kern et al. However, analyses of heparin column flow-through and wash fractions by slot-blot analysis failed to detect AAV-2 particles, suggesting the heparin-binding region was not affected by ligand insertions in our studies.

There was no significant difference in particle titer of mutant viruses compared with wild-type luciferase-encoding virus. Initial studies to determine the transduction efficiency of the mutant capsids was performed in HEK cells. These studies were intended to answer two primary questions: Because amino acid junction — is involved in heparin binding and internalization of the vector, disruption of the motif by inclusion of additional amino acids might affect either heparin binding or infectivity.

Alternatively, the presence of a cellular receptor with binding affinity for genetically incorporated ligands on mutant capsids might augment vector transduction, synergistic to the heparan sulfate pathway. Results, shown in Fig. Interestingly, among the six different capsid mutants, only one m2 , containing the ligand sequence THALWHT, showed significant transduction whereas none of the other mutants m1, m3, m4, m5, and m6 transduced HEK cells.

Having determined the relative transduction efficiency of rAAV-luc containing wild-type or mutant capsids in cells, the next set of experiments was designed to analyze the transduction efficiency of these vectors in a human airway epithelial cell line. Initial studies were performed in Calu-3, a human cell line derived from the serous glandular cells of airway submucosal glands, and typically used to monitor interventions for CF Finkbeiner et al.

Many of these features have been reported previously by us and others for this cell type Augeron et al. Similar to that of HEK cells, transduction efficiency of both wild-type and mutant capsid vectors was determined in the human airway epithelial cell line Calu-3, grown as a monolayer in well tissue culture plates.

Although there was a significant increase in transduction efficiency of the m2 virus over the wild-type virus in cells, the increase found in monolayer Calu-3 cells between the two vectors was highly dramatic. These data suggest that the m2 vector may also use an alternative receptor to internalize in Calu-3 cells, in addition to the HSPG receptor used by unmodified AAV2, and the fact that transduction enhancement with m2 was also observed in cells, albeit to a lower extent compared with Calu-3 cells, suggests that the putative cellular receptor with binding affinity for the m2 ligand THALWHT might be expressed on cells.

Luciferase activity in Calu-3 cells transduced with rAAV-luc containing wild-type and mutant capsids, and the effect of soluble heparin on transduction of wild-type and m2 rAAV-luc in Calu-3 cells. Luciferase activity was determined from cell lysates and expressed as relative light units, normalized to the protein content of each cell lysate. The luciferase assay was done, using cell lysates from individual transductions, and values were normalized to the protein content of each lysate.

Data from studies with m2 virus indicated significant transduction of this vector in HEK and Calu-3 cells. Because the mutant virus was purified to homogeneity by passage through a heparin affinity column, it is likely that the transduction pathway used by the m2 virus could also involve the native HSPG entry mechanism identified for wild-type AAV2. It is also possible that addition of the ligand sequence THAL-WHT could synergize transduction enhancement rather than allowing entry through an entirely different receptor.

To determine whether the m2 virus increases transduction only in the presence of heparin-binding domains or transduces cells entirely through an alternative receptor, independent of the HSPG pathway, heparin-blocking experiments were performed. These data are represented in Fig. Although data from the previously described experiments strongly suggested that the m2 virus is capable of transducing Calu-3 cells in a heparan sulfate-independent manner, a major limitation in the transduction of human airway epithelium in vivo is the lack of virus accessibility to the basolateral sides of well-differentiated cells.

Previous studies with recombinant Ad and AAV have in fact reported transduction through the basolateral surface, where sufficient AAV receptor is present, but not from the apical surface Duan et al. Thus, it was important to determine the ability of m2 virus to transduce human airway epithelial cells on differentiation to polarized epithelium. Airway epithelial cells including Calu-3 form confluent polarized epithelia with transepithelial resistance when grown on a semipermeable support membrane.

The cells also differentiate to form ciliated cells and constitutively express CFTR, features that mimic the actual in vivo physiology in the human airway Foster et al. To test this, Calu-3 cells were differentiated on Transwell filters. Under these conditions, the cells differentiated as monolayers with transepithelial resistance Mathia et al. During this time, Calu-3 cells were transduced with rAAV-luc in wild-type or mutant capsids.

Results are given in Fig. Transduction of rAAV-luc in wild-type or mutant capsids in polarized human airway epithelium. Calu-3 cells were grown in Transwell filters for 7 days to allow polarization. Luciferase activity was measured from cell lysates and expressed as relative light units, normalized to the protein content of each lysate. The results are given in Fig. Free viral particles were removed by washing with PBS.

Luciferase activity from individual samples was normalized to the protein content of each lysate and expressed as relative light units. It was interesting to note that transduction enhancement of the m2 virus was also evident in CBFE cells. Further, in addition to the m2 virus, significant transgene expression was observed from m1 virus, which showed no transduction in either or Calu-3 cells.

Because the ligand incorporated in the m1 mutant, HAIYPRH, has been reported to bind to the human transferrin receptor, it remains possible that these cells may be expressing higher levels of transferrin receptor compared with and Calu-3 cells. However, other mutants m3, m4, m5, and m6 did not show transduction of CFBE cells, as observed in Calu-3 cells.

Monocyte migration across the alveolar epithelium also depends largely on CD47 Rosseau et al. Testing the vector containing the m2 ligand in mouse airway epithelium in vivo did not show a significant increase in gene transfer efficiency. It remains possible that species variation in the target molecule for the incorporated ligand may account for this limitation.

Previous studies on phylogenetic differences in CD47 interactions have reported the existence of significant variation in the binding affinity of this protein for cognate ligands Subramanian et al. Studies have demonstrated that by combining permeability-enhancing compounds with the use of transduction-enhancing compounds, gene transfer through the apical surface of human airway epithelium could be augmented Duan et al.

Whereas a permeability-enhancing compound such as EGTA allows physical display of the vector to target cells, transduction-enhancing compounds act on endosomal processing of the vector. Elucidation of the crystal structure of AAV serotypes that have so far shown promise in transducing airway epithelium, and identification of amenable domains on their capsids for ligand incorporation, should in future allow us to design vectors with augmented transduction for gene therapy of CF. National Center for Biotechnology Information , U.

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The extracellular environment of vascular cells in vivo is complex in its chemical composition, physical properties, and architecture.

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Foxwoods casino sports betting In and Eisenberg took a leave of absence to dr bettinger pforzheim university as deputy director of the law programme being run in China by the German Federal Enterprise for International Cooperation GIZ. Adeno-associated virus type 6 AAV6 vectors mediate efficient transduction of airway free betting tips ht/ft cells in mouse lungs compared with that of AAV2 vectors. The second edition of the manual appeared in fall of Lin X, Helmke B P. Annu Rev Immunol. Depending on the tissue in which cells live, the stiffness of the ECM can strongly vary from very low stiffness e. Initial studies were performed in Calu-3, a human cell line derived from the serous glandular cells of airway submucosal glands, and typically used to monitor interventions for CF Finkbeiner et al.
Bodog sports betting legal in what states It was interesting to note that transduction enhancement of the m2 virus was also evident in CBFE cells. In the first approach, an dr bettinger pforzheim university existing bulk material is structured while in the latter approach single subunits are used to build sports betting ag payout a structured substrate e. The luciferase assay was done, using cell lysates from individual transductions, and values were normalized to the protein content of each lysate. Eisenberg subsequently assumed a position as head of the department for legal affairs, personnel, administration, and PR at Bayern International. There was no significant difference in particle titer of mutant viruses compared with wild-type luciferase-encoding virus. Claudius Eisenberg works to identify such frictions and develop solutions for dealing with them in management practice. Schwartz M A.
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